The role of APOE genotype in the pathogenesis of Alzheimer's disease (AD) remains one of the most critical questions in AD research. Over the past few years, we have pursued an interesting hypothesis that APOE genotype alters normal brain neurochemistry and structure early in life, generating an environment that alters the risk of AD with aging. Our previous studies of APOE knock-in mice have demonstrated that, even in young control mouse brains, APOE genotype affects brain lipid biochemistry, neuronal structure, and spatial learning. In exploring the cause of these various effects, we are now testing the hypothesis that early alterations of brains in APOE4 individuals are due to differences in normal lipid metabolism in astrocytes and neurons. In preliminary studies, we found that APOE isolated from the brain exists in two separate populations: either modified and water soluble, or unmodified and membrane associated. New mass spectrometry approaches allowed us to define that soluble APOE was glycosylated at several sites with several types of glycans; most of the modified APOE in the CNS was not found in the plasma (particularly with unique modification of APOE in its lipid binding domain). In vivo studies showed that APOE4 was less lipidated in CSF lipoproteins compared to APOE2 and APOE3, and that APOE4 mice have altered brain lipid homeostasis. Finally, we found that APOE4-associated phenotypes in mice were rescued by an AD preventative treatment. In this work, we propose to define how brain lipid homeostasis is affected by APOE isoforms, including the newly identified glycoforms. In Aim 1, we will define how APOE modifications alter APOE metabolism, trafficking, and function in the CNS. For these studies, we will identify and quantify new post-translational modifications of APOE across tissues, and develop new astrocyte culture models for expression and modulation of human glyco-APOE isoforms. In Aim 2, we will determine whether APOE4 causes poor lipid efflux in vitro and in vivo, leading to increased activation of LXR targets in mouse and human brains. We will use computer modeling to test the effects of glyco-APOE species on APOE tertiary structure and on APOE binding to CNS lipoproteins. In Aim 3, we will test whether APOE4 causes poor lipid uptake in vitro, leading to changes in neuronal APOE metabolism. We will use glial and neuronal cultures to define how APOE modification changes as APOE travels through CNS compartments. Finally, in Aim 4, we will test whether approaches that prevent AD in mice and humans (anti-inflammatory drugs and exercise/environmental enrichment) affect brain lipid metabolism and brain structure/function. We will test whether our new markers that are affected by the presence of APOE4, including the CNS-specific glycoforms, respond to conditions that reduce AD risk. Thus, this proposal provides new mechanistic insight into the post-translational modification of APOE in the CNS and how the complex set of APOE isoforms in the brain modulate lipid metabolism in ways that could lead to increased risk of AD.